CN114478804A - Fusion peptide targeting pancreatic cancer and application - Google Patents
Fusion peptide targeting pancreatic cancer and application Download PDFInfo
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- CN114478804A CN114478804A CN202210162970.6A CN202210162970A CN114478804A CN 114478804 A CN114478804 A CN 114478804A CN 202210162970 A CN202210162970 A CN 202210162970A CN 114478804 A CN114478804 A CN 114478804A
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- C07K—PEPTIDES
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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Abstract
The invention belongs to the field of biological medicines, and relates to a pancreatic cancer targeted fusion peptide and application thereof. The fusion peptide comprises a membrane-penetrating peptide at the N end and a leader peptide at the C section, wherein the amino acid sequence of the fusion peptide is shown as SEQ ID NO: 1 is shown. According to the biological center rule, the invention researches a transcription factor CTRC nuclear localization signal key sequence, synthesizes a leader peptide for inhibiting the CTRC from entering the nucleus in a targeted manner according to the design, further prepares a fusion peptide, and experiments prove that the fusion peptide can obviously inhibit the growth of pancreatic cancer cells.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a pancreatic cancer targeted fusion peptide and application thereof.
Background
Pancreatic cancer is one of the common malignant tumors in the digestive tract, and is called the king of cancer in the field of tumors. According to the records of the J.Lancet, the five-year survival rate of pancreatic cancer after diagnosis is about 10%, which is one of the worst malignant tumors.
Pancreatic cancer is clinically insidious and atypical, and is a difficult malignancy of the digestive tract to diagnose and treat, with about 90% ductal adenocarcinomas originating from the epithelium of the gland duct. Its morbidity and mortality has increased dramatically in recent years. The early diagnosis rate of pancreatic cancer is low, the operative mortality rate is high, and the cure rate is low. The incidence rate of pancreatic cancer is higher for men than for women, the ratio of men to women is 1.5-2: 1, male patients are more common than for women before menopause, and the incidence rate of postmenopausal women is similar to that of men.
Pancreatic cancer has a high malignancy, a low surgical resection rate, and a poor prognosis. Although surgery remains the primary treatment, combined treatment of pancreatic cancer is required because pancreatic cancer is often found late and the chance of radical cure is lost. To date, as with most tumors, there is no comprehensive treatment regimen that is highly effective and fully applicable. The existing comprehensive treatment still takes surgical treatment as the main part and radiotherapy and chemotherapy as the auxiliary part, and a new method combining biological treatment of immunity, molecules and the like is discussed.
Therefore, the existing treatment means cannot meet the requirement of clinical individualized treatment of pancreatic cancer, and the development of novel treatment targets and medicines is imperative.
Disclosure of Invention
The invention aims to provide a fusion peptide targeting pancreatic cancer, which solves the problems of the existing individual treatment requirements of clinical pancreatic cancer and the development of novel treatment targets and medicines.
The first aspect of the invention is integrated data analysis.
Specifically, by means of integrated analysis of TCGA data, CCLE data, GTEx data and tumor cell growth dependent gene data, the Chymotrypsin (Chymotrypsin C, CTRC) is found to be specifically expressed in pancreatic cancer, and the CTRC is suggested to be a potential target for targeted therapy of pancreatic cancer.
In a second aspect, the invention provides a fusion peptide targeting pancreatic cancer.
Specifically, the fusion peptide comprises a membrane-penetrating peptide at the N terminal and a leader peptide at the C section, and the amino acid sequence of the fusion peptide is shown as SEQ ID NO: 1, and the following components: (RKKRRQRRRRAMWLGIDRPPGIYKKIRKIKNRDYLQYSRSNKR).
Specifically, the penetrating peptide in the present invention may be various penetrating peptides conventional in the art, including but not limited to TAT, Pentratin, Polyarginines, DPV1047, MPG, Pep-1, pVEC, ARF (1-22), BPrPr (1-28), MAP, Transportan, p28, VT5, Bac 7(Bac 1-24), C105Y, PFVYLI, or Pep-7.
Specifically, the fusion peptide of the present invention can be synthesized by a method conventional in the art.
In a third aspect, the invention provides an application of the leader peptide in preparing a pancreatic cancer cell growth inhibitor.
Specifically, the leader peptide has an amino acid sequence shown as SEQ ID NO: 2, (RAMWLGIDRPPGIYKKIRKIKNRDYLQYSRSNKR).
Specifically, according to the biological center rule, the invention researches a key sequence of a CTRC nuclear localization signal, synthesizes a leader peptide for inhibiting the CTRC from entering the nucleus in a targeted mode according to the design, further prepares a fusion peptide, and experiments prove that the fusion peptide can obviously inhibit the growth of pancreatic cancer cells CAPAN-2 and PATU 8988 t.
The invention has the beneficial effects that: provides a fusion peptide targeting pancreatic cancer, can meet the requirement of clinical individualized treatment of pancreatic cancer, and solves the problem of developing novel treatment targets and medicaments for pancreatic cancer treatment.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 shows that the knockout of CTRC targets inhibit the growth of CTRC-highly expressing pancreatic cancer cells.
Fig. 2 shows that knocking down CTRC targeting inhibits growth of CTRC-highly expressing pancreatic cancer cells.
FIG. 3 is a p3 xFLAG-Myc-CMV-25 empty plasmid map.
FIG. 4 shows that the fusion peptide significantly inhibits the growth of pancreatic cancer cells CAPAN-2 on the third and fourth days.
FIG. 5 shows that the fusion peptide significantly inhibited the growth of pancreatic cancer cell PATU 8988t on the third and fourth days.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1: this example demonstrates that intervention in CTRC specifically inhibits growth of CTRC-highly expressing pancreatic cancer cells.
1. Cancer cell growth dependent gene analysis.
1.1 Whole genome knockout library screening data analysis.
Logging in a Depmap portal (https:// deppmap. org/portal /) portal, inputting a gene name 'CTRC', automatically identifying, selecting 'CTRC (ELA 4, CLCR)', selecting a data set 'CRISPR (Depmap 21Q4 Public, Choronos)' on the left side, and clicking 'verification Effects' to download the result, as shown in FIG. 1.
1.2 Whole genome knockdown library screening data analysis.
Logging in a Depmap portal (https:// deppmap. org/portal /) portal, inputting a gene name 'CTRC', automatically identifying, selecting 'CTRC (ELA 4, CLCR)', selecting a data set 'RNAi (Achilles + DRIVE + Marcotte, DEETER 2)', clicking 'Pertturbation Effects', and downloading a result, as shown in FIG. 2.
Each circle in fig. 1 and 2 represents a pancreatic cancer cell line, and the size of the circle indicates high and low CTRC expression; color indicates abrupt change; a cell growth dependent parameter CERES or DEMETER2 value < 0 indicates that CTRC promotes cell growth and >0 indicates that CTRC inhibits cell growth. Red line at-1 is the threshold, left side of red line indicates that CTRC significantly affected cell growth, right side indicates that CTRC did not significantly affect cell growth. As can be seen from fig. 1, the knockdown of CTRC targeting inhibits growth of CTRC-highly expressing pancreatic cancer cells, and as can be seen from fig. 2, the knockdown of CTRC targeting inhibits growth of CTRC-highly expressing pancreatic cancer cells.
Example 2: this example serves to illustrate that the CTRC nuclear localization signal peptide affects CTRC nuclear import.
1. Predicting the CTRC nuclear localization signal sequence.
Logging in an NCBI Protein database (https:// www.ncbi.nlm.nih.gov/Protein), inputting 'CTRC', selecting 'chymotrypsin-C preproprotein [ Homo sapiens ]', and downloading an amino acid sequence of the CTRC Protein; logging in a nuclear localization signal prediction tool NLS _ Mapper (http:// NLS-Mapper. iab. keio. ac. jp/cgi-bin/NLS _ Mapper _ form. cgi), inputting a CTRC amino acid sequence, clicking 'Predict NLS', downloading a result, and predicting and displaying the CTRC nuclear localization signal amino acid sequence by an online tool as follows: ACNLNCQLENGSWEVFGIVSFGSRRGCNTRKKYID are provided.
2. And (5) cloning and constructing.
2.1 obtaining the insert.
The full-length CTRC CCDS region and CTRC nuclear localization signal deletion DNA sequences were synthesized by nivezhi biotechnology limited, su.
2.2 vector digestion.
A50. mu.l digestion system was prepared according to Table 1. Sequentially adding various reagents according to the sequence of a list, lightly blowing and uniformly mixing by using a pipette, carrying out instantaneous centrifugation, and reacting for 3 hours at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band. The p3 xFLAG-Myc-CMV-25 plasmid was purchased from Neptu biosciences, Shanghai, and the map of the empty plasmid is shown in FIG. 3.
Table 1: PCR fragment and vector p3 xFLAG-Myc-CMV-25 recombinant system
2.3 recombinant ligation of the vectors.
The CTRC DNA fragment was directionally cloned into the vector by homologous recombination using the plus One step PCR Cloning Kit (from Novoprotein). A20. mu.L reaction system was prepared according to Table 2 and reacted at 50 ℃ for 10 min.
Table 2: CTRC fragment and vector H15998 recombinant system
2.4 transformation.
Adding 10 μ L of the ligation reaction product into 100 μ L of competent cells, flicking the tube wall, mixing, and standing on ice for 30 min; heat shock is carried out for 90s at 42 ℃, and incubation is carried out for 2min in ice bath; adding 500 μ L LB culture medium, and shake culturing at 37 deg.C for 1 h; taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 h.
2.5 sequencing identification.
And (3) inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing for 12-16h at 37 ℃, and taking a proper amount of bacterial liquid for sequencing and identifying.
3. The cellular immunofluorescence assay detects the effect of nuclear localization signals on the nuclear transport of CTRC.
3.1 cell transfection.
When CAPAN-2 cells in the 12-well plate are converged to 60%, 1.6 mu l of Lipo8000TM and 800ng of plasmid are respectively added into 50 mu l of serum-free culture medium, mixed uniformly, kept stand for 5min at room temperature, added into the 24-well plate, and the culture medium is replaced by fresh culture medium after 8 h.
3.2 Hoechst staining and visualization.
Taking cells cultured by a 12-hole plate, removing a culture medium, adding 500 mu l of Hoechst 33258, and fully covering the cells; continuously culturing the cells for 30 min; discarding the staining solution, washing 3 times with PBS; sealing the anti-fluorescence quenching liquid; and observing the nuclear localization condition of the EGFP-CTRC fusion protein by using a laser confocal microscope. Cell immunofluorescence experiments prove that deletion of a nuclear localization signal NLS inhibits the nuclear transport of the CTRC. The CTRCs of the unloaded and NLS-deleted groups were predominantly localized to the cytoplasm, and the CTRC full-length group showed that CTRCs were predominantly localized to the nucleus.
Example 3: this example is used to demonstrate the effect of the fusion peptides of the invention in inhibiting the growth of pancreatic cancer cells.
1. And (3) synthesizing the fusion peptide.
The sequences of the fusion peptide TAT-CTRC and the reference polypeptide TAT-Contr are shown in Table 3, and the fusion peptide and the reference polypeptide can be obtained by synthesis.
Table 3: fusion peptide and control polypeptide sequence.
Gene | Polypeptide sequence | Size and breadth |
Fusion peptides | RKKRRQRRR-RAMWLGIDRPPGIYKKIRKIKNRDYLQYSRSNKR | 43aa |
Control polypeptide | RKKRRQRRR-PKGRDIFNLSLKYIHLKREKAWEGQELLFKIVAS | 43aa |
2. Cell proliferation assay.
Resuspend 7.0X 10 separately510mg of TAT-CTRC or TAT-Contr are respectively added into each pancreatic cancer CAPAN-2-GFP and PATU 8988t-GFP cell, and the cells are incubated for 1h at 37 ℃; after the culture medium is resuspended, 3000 cells/well are inoculated in a 96-well plate, the plate is photographed at regular time every day for 4 days, the fluorescence intensity is calculated, a growth curve is drawn, and statistical analysis is carried out on SPSS 16.0.
FIG. 4 shows that the fusion peptide significantly inhibits the growth of pancreatic cancer cells CAPAN-2 on the third and fourth days.
FIG. 5 shows that the fusion peptide significantly inhibited the growth of pancreatic cancer cell PATU 8988t on the third and fourth days.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> Jiangsu medical profession college
<120> pancreatic cancer targeted fusion peptide and application thereof
<141> 2022-02-22
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 43
<212> PRT
<213> Artificial Sequence
<400> 1
Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Ala Met Trp Leu Gly Ile
1 5 10 15
Asp Arg Pro Pro Gly Ile Tyr Lys Lys Ile Arg Lys Ile Lys Asn Arg
20 25 30
Asp Tyr Leu Gln Tyr Ser Arg Ser Asn Lys Arg
35 40
<210> 2
<211> 34
<212> PRT
<213> Artificial Sequence
<400> 2
Arg Ala Met Trp Leu Gly Ile Asp Arg Pro Pro Gly Ile Tyr Lys Lys
1 5 10 15
Ile Arg Lys Ile Lys Asn Arg Asp Tyr Leu Gln Tyr Ser Arg Ser Asn
20 25 30
Lys Arg
<210> 3
<211> 43
<212> PRT
<213> Artificial Sequence
<400> 3
Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Lys Gly Arg Asp Ile Phe
1 5 10 15
Asn Leu Ser Leu Lys Tyr Ile His Leu Lys Arg Glu Lys Ala Trp Glu
20 25 30
Gly Gln Glu Leu Leu Phe Lys Ile Val Ala Ser
35 40
Claims (4)
1. A fusion peptide for targeting pancreatic cancer, which comprises an N-terminal cell-penetrating peptide and a C-segment leader peptide, wherein the amino acid sequence of the fusion peptide is shown in SEQ ID NO: 1 is shown.
2. The pancreatic cancer-targeting fusion peptide of claim 1 wherein said cell-penetrating peptide is TAT, penitratin, Polyarginines, DPV1047, MPG, Pep-1, pVEC, ARF (1-22), BPrPr (1-28), MAP, Transportan, p28, VT5, Bac 7(Bac 1-24), C105Y, PFVYLI, or Pep-7.
3. The pancreatic cancer-targeting fusion peptide of claim 1 wherein said leader peptide amino acid sequence is set forth in SEQ ID NO: 2, respectively.
4. The pancreatic cancer-targeting fusion peptide of claim 1, wherein said leader peptide is used in the preparation of a pancreatic cancer cell growth inhibitor.
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